Estimation of blood glucose by Oxidase-peroxidase Method

Estimation of blood glucose by Oxidase-peroxidase Method

Estimation of blood glucose by Oxidase-peroxidase Method

 

Principle of Oxidase-peroxidase Method

Glucose oxidase catalyses the oxidation of glucose to produce hydrogen peroxide and gluconic acid. The hydrogen peroxide, in the presence of enzyme peroxidase is broken down and the oxygen given off reacts with 4-aminophenazone and phenol to give a pink colour.

 

Reagents

Protein precipitant  

• 10 g sodium tungstate, 10 g disodium hydrogen phosphate and 9 g sodium chloride, all dissolved in 800 ml of distilled water. 

• Add 125 ml of 1.0 M HCl to bring the pH to 3.0. Add 1 g phenol and dilute to 1 litre with distilled water. 

• This solution has very long shelf-life at 4°C.

Phosphate buffer,

•  pH 7.0 Dissolve 8.52 g of disodium hydrogen phosphate and 5.44 g of potassium dihydrogen phosphate in 500 ml of distilled water. Adjust the pH and make up to 1 litre. 

Colour reagent 

•  300 ml of phosphate buffer, add 5 ml of Fermcozyme 952 DM (glucose oxidase and peroxidase), 0.3 g phenol, 0.3 g sodium azide and 0.1g 4-aminophenazole. 

• Mix and store at 4°C. It is stable for at least 2 months.

Standard glucose solution  

• (10 mmol/L or 180 mg/dl): Dissolve 1.80 g of glucose in, and make up to, 1 litre with saturated (0.3%) benzoic acid. Prepare the benzoic acid at least 24 hours before use.

Technique

(i) Set up three test tubes as follows:

	Blank	standard	test
Protein precipitant	2.9ml	2.9ml	2.9ml
Distilled water	0.1ml
Working  standard (10 mmol/L0		0.1ml
Test sample			0.1ml

(ii) Mix well and centrifuge at 2500 rpm for 5minutes.

Tubes	1	2	3	4	5	6
Glucose std (ml)	0.0	0.1	0.2	0.3	0.4	0.5
Protein precipitant (ml)	6.0	5.9	5.8	5.7	5.6	5.5
Final conc. Of glucose (mmol/L)	0.0	5.0	10.0	15.0	20.0	25.0

Take a second set of tubes as in (i) above. 

	Blank	Standard	Test
Supernatant	1.0 ml	1.0 ml	1.0 ml
Colour reagent	3.0 ml	3.0 ml	3.0 ml

  

(iv) Mix well and incubate all tubes at 37°C for 10 min. Shake occasionally.

(v) Remove from the water bath, cool and measure the absorbance within 30 minutes. Set the zero with the Blank. Colorimeter: Green filter Spectrophotometer: 515 nm.

Calculations

Alternatively

• a calibration curve can be prepared for the measurement of blood glucose . Dilute the glucose standard as shown in Table below.

• Mix well, remove 1.0 ml from each tube and add 3.0 ml of the colour reagent.

• Mix well, and proceed as steps (iv) and (v) above.

• Plot a graph of absorbance of each standard on vertical axis against its concentration in mmol/L on the horizontal axis.

•  A linear calibration should be obtained as shown in Fig. 3.2.

• The concentration of glucose in the test sample can be obtained from the graph after reading its absorbance.

calibration curve for estimation of glucose

Causes of Abnormal Glucose Levels
Severe hyperglycaemia	Mild/transient hyperglycaemia	Severe hypoglycaemia
1. Diabetes mellitus	1. Severe liver disease	1. Insulinoma
2. Adrenal cortical hyperactivity (Cushing's syndrome)	2. Pancreas disorder	2. Hypopituitarism
3. Acromegaly	3. Pituitary disorder	3. Ectopic insulin production from tumours
4. Obesity	4. Steroid therapy	4. Adrenal cortical insufficiency
	5. Shock 6. Convulsions
	7. Acute stress reaction (Physical or emotional)